克雷伯氏菌产1,3-丙二醇ldhA基因缺失菌株的构建  

Construction a Metabolic Engineering Strain to Produce 1,3-propanediol from Klebsiella pneumoniae by ldhA Gene Deletion Mutation

作　　者：陈利飞[1] 李猛[1] 马春玲[1] 杨建楼 

机构地区：[1]齐鲁工业大学食品与生物工程学院山东省微生物工程重点实验室,济南250353

出　　处：《生物技术通报》2015年第3期121-126,共6页Biotechnology Bulletin

基　　金：济南青年科技明星计划(20090118)

摘　　要：克雷伯氏菌(Klebsiella pneumonia)甘油歧化发酵生产1,3-丙二醇(1,3-PD)的过程中,乳酸是氧化途径最主要的副产物,乳酸的产生和积累,不仅限制了菌体本身的生长,而且严重影响了1,3-丙二醇的转化率。利用λRed重组技术对Klebsiella pneumonia中的酶乳酸脱氢酶基因(ldhA)进行改造。在λRed重组系统作用下,将带有300 bp的线性同源片段ldhA1-Cm-ldh A2与基因组DNA的同源重组,经过抗性筛选和PCR鉴定最终获得了ldhA基因缺失菌株K.pneumonia2-1ΔldhA。经过24 h发酵可知,乳酸最大产出浓度由原来的10.16 g/L降为0.49 g/L,1,3-PD由原来的78.83 g/L增长为85.76 g/L,甘油转化率由60.64%增长到65.97%,提高了5.33%。Production of 1, 3-propanediol from glycerol by Klebsiella pneumoniae is restrained by lactate formation. The increase of lactate in the broth can inhibit cell growth and reduce 1, 3-propanediol conversion rate. The lactate dehydrogenase gene（ldhA）of production lactate by Klebsiella pneumoniae was modified by λRed recombination system. One 300 bp homologous recombinant fragment ：ldh Al-Cm-ldhA2 was constructed for the gene knockout. After resistance experiments, PCR determination, K. pneumonia2-1ΔldhA with ldh A gene knocked out obtained by λRed recombination. After 24 h fermentation, the maximum yield of lactate was reduced to 0.49 g/L from 10.16 g/L and the maximum threonine yield of 1, 3- propanediol was increased to 85.76 g/L from 78.83 g/L, comparing with that of the original strain K. pneumonia2-1. The conversation rate of glycerol was improved to 65.97% from 60.64%, increasing by 5.33%.

关 键 词：克雷伯氏菌 ldhA基因 λRed同源重组 1 3-丙二醇 

分 类 号：Q93[生物学—微生物学]